Novel genes in gonadal development

Abstract: Mammalian sex determination and differentiation can be divided into three major steps. In the first step, which occurs at fertilisation, the genetic sex is established, XX or XY. In the second step, development of the indifferent gonad into either an ovary or a testis takes place. During the third step, translation of gonadal sex into phenotypic sex occurs, resulting in the formation of male or female internal and external genitalia. In the male, this step requires the action of sex hormones such as Anti-Müllerian hormone (Amh), testosterone and dihydrotestosterone. In the absence of testicular hormones, female sexual differentiation occurs. Therefore, the female differentiation is considered as the default pathway. Sry, the sex determining gene on the Y chromosome, is the primary signal for directing the indifferent gonad into the male differentiation pathway. Between 12 and 12.5 dpc the first morphological sign of male differentiation appears, with the formation of testis cords that contain Sertoli- and germ cells. In addition to Sry, several transcription factors controlling sex determination/differentiation have been isolated. These include the Sry-related gene Sox9, two orphan members of the nuclear hormone receptor family, Sf-1 and Dax1, and the Wilms' tumourassociated gene, Wt-1. Although knowledge about the function of these transcription factors is constantly increasing little information is available on downstream events following the activation of these factors. These events include processes such as gonad- mesonephros interactions, migration of cells from the mesonephros into the gonad, Sertoli and Leydig cell differentiation, cross-talk between Sertoli and germ cells and aggregation of cells into testis cords. The aim of this project was to isolate and characterise novel genes involved in gonadal development using the mouse embryonic gonad as a model system. We have developed a modified mRNA differential display method, signal peptide differential display (SPDD), in order to isolate genes involved in morphogenic aspects of testis development and which may act in cell signalling pathways. Using this method, we isolated a novel testis-specific gene named testatin, whivh has homology to protease inhibitors, the cystatins. The restricted expression pattern during cord- formation in the embryonic testis implicates a role for testatin in tissue reorganisation during early testis development. Testatin, together with two other genes, cres and cystatin T, is proposed to belong to a new subgroup of family 2 cystatins. We show differences in spatial and temporal expression patterns of cres, cystatin T and testatin during gonadal development and in adult testis. Genetic studies on individuals that exhibit sex reversal have led to the isolation of genes important to sex development. We have isolated and cloned the human Testatin gene, analyzed its RNA expression in adult and fetal tissues and mapped its chromosomal location. In the hunt for a human phenotype which could be linked to mutations in Testatin, we have sequenced the gene in individuals with disturbed gonadal development. XY individuals with a duplication of the DSS (dosage sensitive sex reversal) locus on the human X chromosome exhibit male-to-female sex reversal. In addition to the DAX1 gene, the DSS region contains the MAGE-B/DAM genes. In an endeavour to isolate mouse homologues to MAGE-B genes, we isolated the Mage-b4 gene from fetal mouse testis cDNA libarary. Mage-b4 expression is confined to the cytoplasm of pre-meiotic germ cells in testis, while in females Mage-b4 is found throughout meiosis. Several factors suggest a growth suppressor role for Mage-b4.